Occurrence and accumulation of potentially infectious viruses in process water and impact of water disinfection practices to minimize viral cross-contamination

Despite accounting for the major causes of foodborne outbreaks, enteric viruses have received comparatively less attention than other foodborne pathogens and thus, virological quality of process water used by the produce industry has received limited attention. As water disinfection is one of the most critical processing steps in vegetable production aimed at preventing cross-contamination, the main purpose of this proposal is to assess the risk of infectious viruses in process water under several scenarios by using viability PCR and correlate their occurrence with the presence of coliphages, which has been suggested as viral indicator. Additionally, this project will investigate the efficacy of the most common disinfection agents used in processing facilities to avoid cross-contamination during washing. Operational conditions and critical parameters will be established for each washing system needed for the inactivation of enteric viruses and coliphages. The validation activities proposed in this project will contribute to get insight if selected disinfectant conditions are capable of effectively controlling the potential presence of human enteric viruses under commercial conditions of process wash water disinfection and the potential use of coliphages as viral indicators. This proposal will contribute to the understanding of enteric viruses as a potential risk to produce-borne outbreaks.

The presence of human enteric viruses like norovirus (NoV), hepatitis A virus (HAV), astroviruses (HAstV) and rotavirus (RV) in irrigation water has extensively been reported. However, the virological quality of process water used by the produce industry has received limited attention. Molecular-based methods have been used to detect human enteric viruses in water samples. However, these methods cannot discriminate between inactivated and potentially infectious viruses. Recent studies have proposed the use of viability markers incorporated into qPCR-based methods for assessing infectivity of enteric viruses in several types of water samples. Bacteriophages infecting enteric bacteria have been suggested as a viral indicators because they mimic viruses better than any other group of indicators. Somatic coliphages and F-specific RNA phages are the major groups investigated for water quality assessment. In this project, the occurrence of potentially infectious viruses will be examined by viability PCR in different process waters from whole and fresh-cut products collected from commercially facilities. Moreover, the analysis of somatic coliphages and F-specific RNA phages will be assessed using plaque assays for the potential used of coliphages as viral indicators. The risk of viruses associated to process water not only depends on their occurrence but also on their resistance to disinfectants currently used by the industry to prevent cross-contamination. As limited information is available on the efficacy of chlorine and non-chlorine based sanitizers against enteric viruses in process water, batch scale experiments and experiments using a dynamic disinfection system will be performed to establish the operational conditions and critical parameters of the washing system needed for the prevention of cross-contamination with a mix of cultivable surrogate viruses (human enteric viruses and coliphages). The implementation of selected operational conditions will be operated in the commercial facilities in collaboration with the five industrial cooperators to evaluate water disinfection practices to minimize viral cross-contamination and validate the operational limits. Our objectives are to 1) detection and quantification of potentially infectious enteric viruses and coliphages in process water used from industrial partners, 2) inactivation studies to evaluate the efficacy of chlorine and non-chlorine based sanitizers on human enteric viruses and coliphages, 3) validation of the stablished water disinfection practices in commercial facilities, 4) correlation between inactivation rates and occurrence of enteric viruses and somatic coliphages and F-specific RNA phages. As a result, the risk of virus contamination by fresh produce process water will be identified. The results of this proposal will contribute to the prevention of viral cross-contamination through water disinfection practices, helping producers and processors to establish the operational standards in process water when using chlorine and non-chlorine based sanitizers.

1. Detection and quantification of potentially infectious enteric viruses and coliphages in process water used from industrial partners 

2. Inactivation studies to evaluate the efficacy of chlorine and non-chlorine based sanitizers on human enteric viruses and coliphages 

3. Validation of the established water disinfection practices for enteric viruses and coliphages in commercial facilities – to be carried out on-line in three disinfectant-produce combinations (i.e., leafy greens and sodium hypochlorite, peppers and PAA and tomatoes and ClO2) 

4. Establishment of coliphages as a suitable indicator of enteric viruses in commercial facilities.

1. Overall, the project provided critical data on (i) levels of coliphages and human enteric viruses in process wash water (PWW) and, (ii) effects on norovirus surrogates, hepatitis A virus (HAV), and MS2 phage of free chlorine (FC), chlorine dioxide (ClO2) and peracetic acid (PAA) treatments at established operational limits for the disinfection of different types of PWW. 

2. Depending on the product, the water ratio, type of product washed in the water, and residual concentration of the sanitizer, the prevalence and concentration of bacteriophages varied significantly. 

3. The study provides industry with important data on (i) current industrial washing practices using FC that are effective for infectious virus and phage control, and (ii) depending on the type of product washed, product/water ratio, and residual concentrations of the sanitizers, the prevalence and concentration of bacteriophages changed significantly. 

4. A key finding of this study is that MS2 inactivation follows similar profiles as norovirus surrogates, so it will facilitate validation in industrial settings. 

5. It has been demonstrated that current operational limits for FC and ClO2 are satisfactory to inactivate human enteric viruses in PWWs and prevent cross-contamination, while higher concentrations would be needed for PAA. 

6. The validation results confirmed the contribution of the high COD (995 mg/L), particularly the high suspended matter present in the PWW of bell peppers supporting microbial aggregates attached to or embedded into the suspended solids, demonstrating the relatively low impact of PAA on water disinfection. 

7. It was not possible to establish a threshold value for any of the tested indicators that could be correlated with a higher probability of the prevalence of human enteric viruses. The absence of crAssphage or bacteriophages cannot be unmistakably associated with the absence of human enteric viruses.